Structures of the ON and OFF states of the membraneous fumarate/Citrate sensor kinases DcuS from E. coli and CitA from Geobacillus thermodenitrifcans

传感器激酶(SK)是细菌对细胞外刺激作出反应的主要机制，导致SK的自动磷酸化。它们的功能包括信号感知、跨膜(TM)信号和信号转导到该激酶。SK的结构域组成和跨膜信号表现出很大的差异。该提案旨在解决DCU/CITA家族SK(富马酸和柠檬酸感知)在开启和关闭状态下的结构，并了解跨膜和细胞内信号转导中结构域的重构。DCU/CITA包含跨膜信号SK的古典型结构域组成。最近，从大量的TM感受器激酶中，NarQ硝酸盐SK的结构已经被解决，其形式是胞质中的GAF和KK结构域被耗尽。NarQ代表一种独特的SK，其结构域组成与DCU/CITA有很大不同，很可能代表一种不同的信号转导模式。大肠杆菌的DCU和嗜热地杆菌的CITA都是同源二聚体，分别由富马酸或柠檬酸结合的胞外PASP(Per Arnt Sim)结构域、两个TM螺旋(TM1、TM2)、细胞质PASC和激酶域组成。根据分析系统中蛋白质的可用性，用DCUS或CITA进行互补研究。聚天冬氨酸和聚天冬氨酸的结构已经在分离的和膜完整的结构中被解决，在开和关状态。结构、遗传和生化研究表明，PASP在底物结合上紧凑，触发TM2活塞式运动，负责跨膜信号转导。PASC在α-螺旋二聚部位的结构重排中响应TM2的移位。这项工作是高分辨率分析的基础，并对跨膜信号转导过程中结构域的结构重排进行了综合观察。工作将集中在(I)TM区的结构重排，TM2的活塞式运动可能与TM2的剪刀式运动相结合，(Ii)PASC在信号传递和对激酶域的控制过程中的结构重排。为了获得完整的视图和高分辨率，将使用结构(核磁共振和结晶；AG Griesinger)、生化、遗传(AG Unden)和生物物理(AG Hinderberger，Griesinger)方法。准备工作确立了研究的所有结构和方法，我们假设DCU/CITA是通过结构生物学、生物化学和生物物理学的结合方法获得高结构和功能分辨率的极少数跨膜和细胞质信号可及的SK之一。

Sensor kinases (SK) represent a major device of bacteria for responding to extracellular stimuli, resulting in autophosphorylation of the SK. Their function includes signal perception, transmembrane (TM) signaling, and signal transmission to the kinase. Domain composition and transmembrane signaling of the SK show broad variation. The proposal aims at solving the structure of a SK of the DcuS/CitA family (fumarate and citrate sensing) in the ON and the OFF state, and to understand restructuring of the domains in transmembrane and intracellular signaling. DcuS/CitA contain an archaetypical domain composition for transmembrane signaling SK. From the vast number of TM sensor kinases, the structure of the NarQ nitrate SK has been solved in a form depleted of the cytosolic GAF and kinase domains recently. NarQ represents a unique SK with a domain composition largely different from DcuS/CitA and stands very likely for a different mode of signal transduction. DcuS of E. coli and the closely related CitA of the thermophilic Geobacillus thermodenitrificans are homodimers each, composed of an extracytoplasmic PASP (Per ARNT Sim) domain for fumarate or citrate binding, two TM helices (TM1, TM2), a cytoplasmic PASC and the kinase domain. Studies are performed with DcuS or CitA complementarily according to availability of the proteins for the analytic systems. Structures of PASP and PASC have been solved in isolated and membrane-integral constructs in the ON and OFF states. Structural, genetic and biochemical studies show a compaction of PASP upon substrate binding that triggers a piston-like movement of TM2 which is responsible for transmembrane signaling. PASC responds to the shift of TM2 in a structural rearrangement at an alpha-helical dimerization site. This work is the basis for high resolution analysis and an integrated view of the structural rearrangements in the domains during transmembrane signaling. Work will concentrate on (i) on the structural rearrangements in the TM region with the piston-like movement of TM2 that may be combined with a scissors movement of TM2, (ii) the structural rearrangement in PASc during signal transmission and control of the kinase domain. For a complete view and high resolution, structural (NMR and crystallization; AG Griesinger), biochemical, genetic (AG Unden) and biophysical (AG Hinderberger, Griesinger) methods will be applied. Preparatory work established all constructs and the methods for the study, and we presume that DcuS/CitA represent one of the very few SK whose transmembrane and cytoplasmic signaling is within reach at high structural and functional resolution by a combined approach from structural biology, biochemistry and biophysics.